title: Improving the activity of cytochrome P450 BM-3 catalyzing indole hydroxylation by directed evolution.
abstract: Cytochrome P450 BM-3 (A74G/F87V/L188Q) could catalyze indole to produce indigo. To further improve this capability, random mutagenesis was performed on the heme domain of P450 BM-3 (A74G/F87V/L188Q) with error-prone PCR. A single mutant V445A was selected out from the error-prone library and exhibited the highest specific activity toward indole among the mutants obtained. The kinetic parameters of V445A were also highly improved. Compared with the parent enzyme, the turnover rate (k cat) of V445A was increased by 7.5 times, while its K m value decreased by 9.2 %. Consequently, the catalytic efficiency (k cat/K m) of V445A was raised to 8.2 times than that of the parent enzyme. Moreover, alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Three-dimensional structure analysis was also used to rationalize the effect on the enzyme properties of the mutation. This study showed that random mutagenesis was efficient to identify mutants with potential values in industry and increased our insight into P450 BM-3.
authors.affiliation: Institute of Bioengineering, Zhejiang University, Hangzhou, 310027, Zhejiang, People's Republic of China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Pengpai
authors.forename: Hu
authors.initials: H
authors.lastname: Sheng
authors.forename: Mei
authors.initials: M
authors.lastname: Lehe
authors.forename: Lei
authors.initials: L
authors.lastname: Yinlin
authors.forename: Jin
authors.initials: J
authors.lastname: Zhihua
authors.forename: Hu
authors.initials: H
authors.lastname: Guixiang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004579:Electron Transport; D015723:Gene Library; D006900:Hydroxylation; D007211:Indoles / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D007211:Indoles; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases